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TCM (central memory T cell) induced by peripheral blood mononuclear cells, and application

An immune cell and memory technology, applied in the application field of memory immune cells (TCM), can solve the problems of poor memory T cell proliferation ability, difficult long-term survival of immune cells, immune incompetence, etc., and achieve anti-tumor immunotherapy Flexible and variable, the effect of solving the problems of poor T cell proliferation and immune incompetence

Inactive Publication Date: 2018-12-25
路春光
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of long-term survival of immune cells in vivo and lack of targeting in adoptive cell therapy technology; this application provides a method for isolating and purifying memory T cells from cryopreserved peripheral blood mononuclear cells (PBMC). TCM), TCM induction method with high activation rate, and then transfect tumor-specific TCR gene into autologous TCM, so that immune cell therapy has long-acting and targeted
Solve the problem of poor proliferation ability and immune incompetence of memory T cells in tumor patients

Method used

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  • TCM (central memory T cell) induced by peripheral blood mononuclear cells, and application
  • TCM (central memory T cell) induced by peripheral blood mononuclear cells, and application
  • TCM (central memory T cell) induced by peripheral blood mononuclear cells, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Isolation and cryopreservation of PBMC cells:

[0043]a. Separation of PBMC cells: Peripheral blood is divided evenly into 50mL centrifuge tubes with an electric aspirator according to no more than 35mL per tube, 931 g, centrifuged for 8min, and the upper layer plasma is sucked into 50mL centrifuge tubes, and 1.5mL is put into EP tubes Medium, labeled, and stored at -80°C. Dilute the blood cells with NA so that the ratio of blood cells: NA is 1:1, mix evenly and slowly add to the corresponding upper layer of liquid A to form a complete interface. 596 g, centrifuged for 20 min. Visible stratification. Aspirate and discard the supernatant of the first layer, and carefully and gently aspirate the second layer of white cell layer into different clean 50mL centrifuge tubes. Add NA to each tube respectively, dilute and mix, and dilute to 40mL / tube. 931 g, centrifuged for 8 minutes. Discard the supernatant and add NA to resuspend the cells. The cell suspension was col...

Embodiment 2

[0058] On the basis of Example 1, the TCM cells were sorted and purified, and induced and transfected in vitro, as follows

[0059] 1. Magnetic bead sorting of T cell subsets:

[0060] (1) Cell count.

[0061] (2) Prepare a single cell suspension (filtered with a 30-pore nylon mesh).

[0062] (3) Add buffer to wash cells, centrifuge at 1500rpm, 5min (4-8°C)

[0063] (4) Add 80ul / 107 cells of buffer.

[0064] (5) Add 20ul / 107 cells of antibody-coated magnetic beads.

[0065] (6) Incubate at 4-8°C for 10 minutes.

[0066] (7) Add 1ml / 107 cells of buffer to wash the cells and centrifuge at 1500rpm for 5min (4-8°C).

[0067] (8) Add 500ul / 107 cells of buffer to resuspend the cells.

[0068] (9) Prepare the separation column and wash the MS column with 500ul of buffer.

[0069] (10) Carefully add the cell suspension to the bottom surface of the sorting column.

[0070] (11) Rinse the column with 500ul of buffer solution (add new liquid each time there is no residual liquid)...

Embodiment 3

[0084] On the basis of embodiment 2 formula a, carry out the selection of induction formula

[0085] On the first day after isolation, complete initial stimulation with 1000IU / ml IFN-γ, anti-human 130ng / ml CD3 monoclonal antibody, anti-human 110ng / ml CD28 monoclonal antibody, recombinant human 1000IU / ml IL-2, 120u / mL IL-1α . Half of the medium was changed every 2 days, and recombinant human IL-2 (final concentration of 1000 IU / ml), 800 u / mL IL-7 and 800 u / mL recombinant human IL-15 were added.

[0086] The isolated PBMCs were resuspended with X-VIVO15, and the initial stimulation was completed with anti-human 130ng / mL CD3 monoclonal antibody, 110ng / mL anti-human CD28 monoclonal antibody and 1000u / mL recombinant human IL-2. The medium was changed in half every 2 days, and 1000 u / mL recombinant human IL-2 and 800 u / mL recombinant human IL-15 were added. On the basis of traditional TCM cell culture, IFN-γ, IL-1α and recombinant human cytokine IL-7 were added.

[0087] The expe...

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Abstract

The invention relates to the technical field of targeted therapy of immune cells, in particular to a TCM (central memory T cell) obtained through induced differentiation of human peripheral blood mononuclear cells. The basis is provided for application of the TCM induced by the frozen peripheral blood mononuclear cells to clinical tumor treatment, the cell can be resuscitated at any time accordingto treatment plans of patients with tumors, and the problems of poor proliferation capability and immune incompetence of T cells of the patients with the tumors are solved; currently, the reinfused immune cells, especially cloning effector T cells, are difficult to survive in the long-term, and the clinical curative effect of the adoptive cell therapy is affected greatly; the TCM has a long-lasting memory, and is transfected through a TCR gene to achieve TCM cell targeting.

Description

technical field [0001] The present invention relates to the technical field of immune cell targeted therapy, in particular to human peripheral blood mononuclear cells induced to differentiate into memory immune cells (TCM), and also relates to the application of memory immune cells (TCM). Background technique [0002] Adoptive cell therapy technology provides an effective method for breaking the central and peripheral immune tolerance of tumor patients, restoring or enhancing their anti-tumor immune function. However, there are still many problems to be solved, including: 1) how to obtain a sufficient number of tumor-reactive T cells is the most critical factor in determining the effect of adoptive cell therapy; 2) the current reinfusion of immune cells, especially cloned CD8+ It is difficult for T cells to survive for a long time in the body, which greatly affects the clinical efficacy of adoptive cell therapy. [0003] Memory T cells can be rapidly activated to exert immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N15/867A61K35/17A61P35/00A61P31/12A61P31/18A61P31/06
CPCA61K35/17A61P31/06A61P31/12A61P31/18A61P35/00C07K14/7051C12N5/0636C12N5/0638C12N15/86C12N2501/2301C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/24C12N2501/51C12N2501/515C12N2506/11C12N2510/00C12N2740/10043
Inventor 路春光
Owner 路春光
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